This invention relates in part to improvements directed to use of RNA interference (RNAi) technology that exploits a newly identified small RNA biogenesis pathway.
Traditional RNAi technology in mammals takes advantage of the canonical microRNA (miRNA) pathway. Starting with PolII transcribed precursor RNAs, the biogenesis pathway involves two steps: DROSHA/DGCR8 cleaves the precursor transcript into a short hairpin RNA that is exported into the cytoplasm and then processed by DICER RNAseIII enzyme to yield a mature small (21-22 nt) RNA duplex, that is then loaded into one of four Argonaute proteins (AGO1 through AGO4) to form an active RNA Induced Silencing Complex (RISC). The conventional endogenous RNAi pathway therefore comprises three RNA intermediates: a long, largely single-stranded primary miRNA transcript (pri-mRNA); a precursor miRNA transcript having a stem-and-loop structure and derived from the pri-mRNA (pre-miRNA); and a mature miRNA.
Argonaute proteins are the key effectors of small RNA-mediated regulatory pathways that modulate gene expression, regulate chromosome structure and function, and provide an innate immune defense against viruses and transposons (Hutvagner, G. & Simard, M. J. Nat Rev Mol Cell Biol 9, 22-32 (2008)). The structure of Ago proteins is well conserved, consisting of an amino-terminal domain, the mid domain, and their signature PAZ and Piwi domains. Structure-function relationships in this family are becoming increasingly well understood (Joshua-Tor, L. Cold Spring Harb Symp Quant Biol 71, 67-72 (2006)). The PAZ and Mid domains help to anchor the small RNA guide, with PAZ binding the 3′ end using a series of conserved aromatic residues and the Mid domain providing a binding pocket for the 5′ end. The Piwi domain contains an RNAse H motif that was cryptic in the primary sequence but easily recognizable in the tertiary structure. Loading of a highly complementary target into an Ago brings the scissile phosphate, opposite nucleotides 10 and 11 of the small RNA guide, into the enzyme active site, allowing cleavage of the RNA to leave 5′ P and 3′ OH termini (Elbashir, S. et al. Genes Dev 15, 188-200 (2001), Elbashir, S. M., et al. EMBO J 20, 6877-88 (2001), Yuan, Y. R. et al. Mol Cell 19, 405-19 (2005), Martinez, J. & Tuschl, T. Genes Dev 18, 975-80 (2004), Schwarz, D. S., et al. Curr Biol 14, 787-91 (2004)).
Ago proteins can be divided into three clades. The Piwi clade is animal specific, and forms part of an elegant innate immune system that controls the activity of mobile genetic elements (Malone, C. D. & Hannon, G. J. Cell 136, 656-68 (2009)). The Wago clade is specific to worms and acts in a variety of different biological processes (Yigit, E. et al. Cell 127, 747-57 (2006)). The Ago clade is defined by similarity to Arabidopsis Ago1 (Bohmert, K. et al. EMBO J 17, 170-80 (1998)). Ago-clade proteins are found in both plants and animals where one unifying thread is their role in gene regulation. In plants, some Ago family members bind to microRNAs and are directed thereby to recognize and cleave complementary target mRNAs (Baumberger, N. & Baulcombe, D. C. Proc Natl Acad Sci USA 102, 11928-33 (2005), Qi, Y., Denli, A. M. & Hannon, G. J. Mol Cell 19, 421-8 (2005)).
Animal microRNAs function differently from their plant counterparts, with nearly all microRNA-target interactions providing insufficient complementarity to properly orient the scissile phosphate for cleavage. Here, target recognition relies mainly on a “seed” sequence corresponding to miRNA nucleotides (Joshua-Tor, L. Cold Spring Harb Symp Quant Biol 71, 67-72 (2006), Malone, C. D. & Hannon, G. J. Cell 136, 656-68 (2009)). While pairing of the target to other parts of the miRNA can contribute to recognition, seed pairing appears to be the dominant factor in determining regulation (Yekta, S. et al. Science 304, 594-6 (2004)). A very few extensive microRNA-target interactions can lead to target cleavage in mammals (Davis, E. et al. Curr Biol 15, 743-9 (2005), Harfe, B. D. et al., Proc Natl Acad Sci USA 102, 10898-903 (2005)). However, none of these has yet been shown to be critical for target regulation (Sekita, Y. et al. Nat Genet 40, 243-8 (2008), Hornstein, E. et al. Nature 438, 671-4 (2005), Tolia, N. H. & Joshua-Tor, L. Nat Chem Biol 3, 36-43 (2007)).
Despite the fact that animal microRNAs regulate targets without Ago-mediated cleavage, the Argonaute catalytic center is deeply conserved. This consists of a catalytic DDH triad that serves as a metal coordinating site (Liu, J. et al. Science 305, 1437-41 (2004)). Of the four Ago-clade proteins in mammals, only Ago2 has retained both the DDH motif and demonstrable endonuclease activity (Rivas, F. V. et al. Nat Struct Mol Biol 12, 340-9 (2005), Song, J. et al. Science 305, 1434-7 (2004), Azuma-Mukai, A. et al. Proc Natl Acad Sci USA 105, 7964-9 (2008)). Ago1, Ago3, and Ago4 are linked within a single ˜190 kb locus and have lost catalytic competence. An analysis of Ago2 mutant cells has indicated that proteins encoded by the Ago 1/3/4 locus can support miRNA-mediated silencing (Rivas, F. V. et al. Nat Struct Mol Biol 12, 340-9 (2005)). This leaves us without a clear explanation for the maintenance of a catalytically competent Ago family member, since miRNAs are the exclusive partners of these proteins in almost all cell types (Babiarz, J. E., Ruby, J. G., Wang, Y., Bartel, D. P. & Blelloch, R., Genes Dev 22, 2773-85 (2008); Ender, C. et al. Mol Cell 32, 519-28 (2008) Tam, O. C. et al. Nature, 453:534-538 (2008); Kaneda, M. et al., Epigenetics Chromatin, 2:9 (2009)).